Methods of treating pd-l1 expressing cancer

ABSTRACT

Described herein are methods, formulations and kits for treating a patient with cancer with nanoparticle complexes comprising a carrier protein, a binding agent and paclitaxel and optionally co-treated with an anti-PD-L1 antibody.

FIELD OF THE INVENTION

This disclosure relates to novel methods and kits for treating cancer byadministering nanoparticle complexes comprising a carrier protein, abinding agent that binds specifically to PD-L1, and paclitaxel. Themethod may also comprise pretreating a patient suffering from a cancercomprising cancer cells that express PD-L1 with anti-PD-L1 antibodyalone, prior to, concurrently with or after administering thenanoparticle complexes.

STATE OF THE ART

Cancers cells employ a variety of means to escape immune surveillanceand thereby continue to proliferate and/or metastasize. For example,many cancer cell types express or overexpress PD-L1 (programmed celldeath ligand 1) (B7-H1), the principal ligand of program cell deathprotein 1 (PD-1). PD-1 is a cell surface receptor on T lymphocytes andis expressed upon activation in mature hematopoietic cells such as T andB cells, NKT cells and monocytes after prolonged antigen exposure(Ishida et al., 1992. EMBO J. 11:3887). Expression of PD-1 and PD-L1 inthe tumor microenvironment appears to be a major resistance mechanism toescape immune surveillance. It is hypothesized that PD-L1 binding toPD-1 on T-cells suppresses effector anti-tumor T-cell activity andfacilitates immune evasion.

There are several clinical trials that use monoclonal antibodies thateither bind to PD-1 or PD-L1 in the treatment of cancers, including lungcancer, bladder cancer, kidney cancer, hematological cancers, breastcancer, colorectal cancer, melanoma and solid cancers. Anti-PD-1antibodies known in the art include, e.g., Nivolumab(BMS-936558/MDX-1106/ONO-4538; Bristol Myers Squibb), PDR001 (Novartis),and Pembrolizumab (MK-3475) (Merck Sharp & Dohme); anti-PD-L1 antibodiesknown in the art include, e.g., BMS-936559/MDX-1105 (Bristol MyersSquibb), Atezolizumab (MPDL3280A, Genetech/Roche), MeDI4736 (durvalumab;MedImmune/AstraZeneca), MSB00100718C (avelumab; EMD Serono) (see, e.g.,Philips and Atkins “Therapeutic uses of anti-PD-1 and anti-PD-L1antibodies” International Immunology Vol. 27(1) pp: 39-46).

Despite the antitumor activity of antibodies targeting the PD-1:PD-L1pathway, resistance to these therapies has been increasingly observed(see, e.g., Lussier et al. J. Immunotherapy of Cancer, 2015, 3:21 andKoyama et al., Nature Communications, 2016 7:1-9 (Published online 17Feb. 2016)). Thus there remains a need in the art to improve theefficacy of cancer therapeutics.

SUMMARY

Described herein are methods for treating a patient suffering from acancer having cancer cells that express a programed cell death ligand 1(PD-L1), particularly e.g., a PD-L1-expressing cancer cells that havebecome resistant to immunotherapy with anti-PD-L1 antibodies. The methodcomprises, or consists essentially of, administering, to a subject inneed thereof, e.g., a mammal having cancer cells that express oroverexpress PD-L1, a composition comprising a therapeutic amount ofnanoparticle complexes comprising (a) a carrier protein, (b) aneffective amount of a binding agent having a PD-L1 binding portion thatbinds to PD-L1 so as to provide directional guidance to the nanoparticlecomplexes to the cancer cells and (c) an effective amount of paclitaxel.

In one embodiment, the average diameter of the complexes is between 0.1and 0.9 m. The binding agent may be an anti-PD-L1 antibody, e.g.atezolizumab. The mammal can be a human. The PD-L1-expressing cancercells may be, e.g., melanoma, renal cell carcinoma, non-small cell lungcarcinoma, head and neck squamous cell carcinoma, colorectal cancer,Merkel cell carcinoma, ovarian cancer, bladder cancer and advanced solidtumors.

The carrier protein/paclitaxel/binding agent nanoparticle complexes canbe ABRAXANE®/anti-PD-L1 antibody complexes. ABRAXANE® is available fromCelgene Corp. and is a nanoparticle formulation that combines paclitaxelwith human albumin. The carrier protein (e.g.,albumin)/paclitaxel/antibody nanoparticle complexes, or a compositioncomprising the complexes, can further comprise an alkylating agent. Thealkylating agent can be a platinum compound. The platinum compound canbe carboplatin. The anti-PD-L1 antibodies can be humanized antibodies.The anti-PD-L1 antibodies can be chimeric antibodies. The compositioncan be administered by injection.

In one embodiment, the PD-L1-expressing cancer cells are resistant toimmunotherapy with anti-PD-L1 antibodies.

In an embodiment of the methods described herein, the complexes areadministered in an amount sufficient to deliver a therapeuticallyeffective amount of the paclitaxel.

The anti-PD-L1 antibody suitable for use in the inventions describedherein includes Atezolizumab (TECENTRIQ™, Genentech, Inc. A Member ofthe Roche Group) or a biosimilar version thereof. In some embodiments,the anti-PD-L1 antibody is BMS-936559/MDX-1105 (Bristol Myers Squibb),Atezolizumab (MPDL3280A, Genetech/Roche), MeDI4736 (durvalumab;MedImmune/AstraZeneca), or MSB00100718C (avelumab; EMD Serono).

In an embodiment of the methods described herein, the target of thecancer cells are cells of a solid cancer.

In an embodiment of the methods described herein, the carrierprotein/paclitaxel/PD-L1 binding agent nanoparticle complexes, e.g.,albumin/paclitaxel/anti-PD-L1 antibody nanoparticle complexes, arelyophilized and may be reconstituted for administration to a subject inneed thereof.

An embodiment of the invention includes a method for increasing theduration of uptake of a chemotherapeutic agent by a tumor that expressesPD-L1. The method includes administering the chemotherapeutic agent in ananoparticle complex comprising a carrier protein, the chemotherapeuticagent (e.g., paclitaxel) and a PD-L1 binding agent, e.g., an antibodythat specifically binds to PD-L1, wherein the binding agent providesdirectional guidance to the nanoparticle complex to the tumor.

As described herein, in vitro mixing of albumin-containing nanoparticles(e.g., ABRAXANE® nanoparticles) and antibodies (e.g., anti-PD-L1antibodies, such as Atezolizumab) can result in the formation ofmacromolecular complexes, the characteristics of which (e.g., size,antibody content, or chemotherapeutic drug content) can be customizeddepending on need. In some cases, such macromolecular complexes canretain antibody mediated target binding specificity, can retain orexhibit enhanced chemotherapeutic tumor cell cytotoxicity, and canexhibit no additional toxicity beyond that of ABRAXANE® nanoparticlesalone. As also described herein, contacting ABRAXANE® with an anti-PD-L1antibody (e.g., Atezolizumab) prior to administration to a human (e.g.,a human cancer patient wherein the cancer expresses or overexpressesPD-L1) can result in a complex that, when administered as a complex, hasan increased ability to treat a cancer as compared to a treatmentregimen that includes administering ABRAXANE® and the anti-PD-L1antibody separately in a manner that does not form ABRAXANE®/anti-PD-L1antibody complexes. The methods and materials provided herein can beused to increase the progression-free survival rate in cancer patients.Increasing progression-free survival can allow cancer patients to livelonger. Thus the methods and materials provided herein can be used toincrease the overall survival rate in cancer patients.

Also described herein are methods for treating a cancer, preferably acancer comprising cancer cells expressing programed cell death ligand 1(PD-L1), by administering to a patient an anti-PD-L1 antibody andnanoparticle complexes, which complexes comprise a carrier protein,paclitaxel and a binding agent that specifically binds to PD-L1, e.g.,an anti-PD-L1 antibody. As described herein, the treatment of thesubject having a cancer, e.g., a cancer that expresses PD-L1, oroverexpresses PD-L1, with the PD-L1 antibody in combination with suchnanoparticle complexes increases the therapeutic efficacy of thecomplexes. Preferably, such anti-PD-L1 antibodies are administered priorto treatment with such nanoparticle complexes. Accordingly, an aspectprovided herein is a method for treating a patient suffering from acancer, e.g., a cancer which expresses or overexpresses PD-L1, whereinthe patient is treated with a sub-therapeutic amount of an anti-PD-L1antibody and a therapeutic amount of nanoparticle complexes comprisingthe carrier, paclitaxel, and anti-PD-L1 antibody. The administration ofthe sub-therapeutic amount of the anti-PD-L1 antibody is such that itenhances the efficacy of the nanoparticle complexes. Without wishing tobe bound by any theory, it is contemplated that administration of asub-therapeutic amount of the anti-PD-L1 antibody enhances thetherapeutic efficacy of the nanoparticle complexes by binding tonon-tumor-bound PD-L1 in the body. Treatment with a sub-therapeuticamount of anti-PD-L1 antibody may allow for greater targeting of thenanoparticle complexes to the tumor, decrease the amounts of the carrierprotein/paclitaxel/antibody complexes administered to a patientnecessary to achieve a desired effect, or both.

In another aspect, provided herein are methods for enhancing theefficacy of carrier protein/paclitaxel/anti-PD-L1 antibody nanoparticlecomplexes by administering the complexes about 0.5 to 48 hours afterpretreatment of a patient with a sub-therapeutic amount of anti-PD-L1antibody. Preferably, such nanoparticle complexes are administered about24 hours after the sub-therapeutic amount of anti-PD-L1 antibody.

In another aspect, provided herein are methods for enhancing thetherapeutic outcome in a patient suffering from a cancer, e.g., a cancerexpressing or overexpressing PD-L1, by treating the patient with asub-therapeutic amount of an anti-PD-L1 antibody (e.g., an uncomplexedanti-PD-L1 antibody, e.g. not bound to a carrier protein/paclitaxelcomplex) and co-treating the patients with an effective amount ofnanoparticle complexes comprising albumin, paclitaxel, and anti-PD-L1antibodies wherein the complexes can bind to PD-L1. In one embodiment,the antibodies are arranged on the surface of the complexes.

In another aspect, provided herein are methods for enhancing thetherapeutic outcome in a patient suffering from a cancer, e.g., a cancerexpressing or overexpressing PD-L1, by treating the patient with asub-therapeutic amount of the anti-PD-L1 antibody prior to anysubsequent treatment with the nanoparticle complexes comprising carrierprotein, paclitaxel, and anti-PD-L1 antibodies, wherein the complexesbind to PD-L1. In one embodiment, the antibodies are arranged on thesurface of the complexes.

In an embodiment of this invention, the methods described herein areadministered to a subject who has a cancer comprising cells that expressPD-L1 but which cancer is resistant to immunotherapy with anti-PD-L1antibodies that are not in complex with nanoparticles comprising acarrier protein-bound chemotherapeutic, e.g., an albuminbound-paclitaxel nanoparticle, e.g., ABRAXANE®.

Examples of cancer cells known to express PD-L1 and thus suitable fortreatment with the methods disclosed herein include but are not limitedto melanoma, renal cell carcinoma, non-small cell lung carcinoma, headand neck squamous cell carcinoma, colorectal cancer, Merkel cellcarcinoma, ovarian cancer, bladder cancer, hematologic cancers, andother solid cancers, which cancers express PD-L1.

In an embodiment, the anti-PD-L1 antibody is atezolizumab or abiosimilar version thereof. Atezolizumab (trade name TECENTRIQ™) is afully humanized, Fc-modified monoclonal antibody of IgG1 isotype againstPD-L1. Other anti-PD-L1 antibodies include MDX-1105, a fully humanmonoclonal antibody that binds to PD-L1; Avelumab (MSB0010718C, MerckKGaA, Darmstadt, Germany & Pfizer), a fully human monoclonal PD-L1antibody of isotype IgG1; and Durvalumab (MedImmune/AstraZeneca), an Fcoptimized anti-PD-L1 mAb.

In one embodiment, the sub-therapeutic amount of anti-PD-L1 antibody isselected from an amount consisting of about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55% or about 60% of the therapeutic dosage ofanti-PD-L1 antibody. It is contemplated that administration of thesub-therapeutic amount of anti-PD-L1 antibody preferentially blockscirculating PD-L1 with minimal blocking of PD-L1 associated with atumor. In some embodiments, the sub-therapeutic amount of anti-PD-L1 tobe administered to the patient is determined by analyzing the level ofcirculating PD-L1 in the blood.

In one embodiment, the sub-therapeutic amount of anti-PD-L1 antibody isadministered from between about 30 minutes to about 48 hours prior toadministration of the albumin/paclitaxel/anti-PD-L1 antibodynanoparticle complexes.

In other aspects provided herein are unit-dose formulations of ananti-PD-L1 antibody, for example, atezolizumab or a biosimilar versionthereof, which formulation comprises from about 1% to about 60% of atherapeutic dose of the antibody wherein the formulation is packaged soas to be administered as a unit dose.

In some embodiments, the formulation of anti-PD-L1 antibodies comprisesfrom about 5% to about 20% of a therapeutic dose of atezolizumab or abiosimilar version thereof. The therapeutic dose for atezolizumab, e.g.for locally advanced or metastatic urothelial carcinoma, is recited inthe prescribing information. The therapeutic dose is 1200 mg andpreferably a subtherapeutic dose ranges from 5% to 20% of thetherapeutic dose. In such a preferred embodiment, such a subtherapeuticdose would range from 60 mg to 240 mg, more preferably from 120 mg to160 mg.

In other aspects, provided herein are kits comprising: (a) an amount ofan albumin/paclitaxel/anti-PD-L1 antibody complexes, (b) a unit dose ofa sub-therapeutic amount of anti-PD-L1 antibody, and optionally (c)instructions for use.

In one embodiment, the carrier-bound paclitaxel (e.g.,albumin-paclitaxel, e.g., ABRAXANE®)/anti-PD-L1 antibody complexes ofthe kits are lyophilized. The lyophilized complexes may be reconstitutedin an aqueous solution prior to administration. The aqueous solutionmaybe a sterile aqueous solution or the reconstituted aqueous solutionmay be filtered sterilized through e.g., a 0.2 or 0.22 μm filter.

An embodiment of the invention includes a method for increasing theduration of tumor uptake of a chemotherapeutic agent by administeringthe chemotherapeutic agent in a nanoparticle complex comprising acarrier protein, paclitaxel, the chemotherapeutic agent and a PD-L1binding agent, e.g. an anti-PD-L1 antibody, the PD-L1 binding agentproviding directional guidance to the nanoparticle complex to the tumor.In some embodiments, the subject receives a subtherapeutic amount of theanti-PD-L1 antibody prior to or concurrently with such nanoparticlecomplexes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are representative only of the invention and arenot intended as a limitation. For the sake of consistency, nanoparticlesusing ABRAXANE® and atezolizumab employ the acronym “AA” and the numberafter AA such as AA130 is meant to confer the average particle size ofthese nanoparticles (in nanometers, based on Malvern Nanosightanalysis).

FIG. 1 shows the binding affinity between atezolizumab and ABX. The Kdwas determined to be 1.462×10⁻⁹. Biolayer interferometry (BLItz) (ForteBioscience) was performed using streptavidin probes.

FIG. 2A shows the particle size distribution for ABX alone (average sizeof 90 nm) and ABX-atezolizumab nanoparticles (AA; average size of 129nm), as determined by Mastersizer NS300. FIG. 2B is a photograph of theABX-atezolizumab nanoparticles from FIG. 8A.

FIGS. 3A-3E show flow cytometry of ABX-atezolizumab nanoparticles(AA130) competing with labeled anti-PD-L1 antibody for binding to aPD-L1 positive human melanoma cell line, C8161. C8161 cells werepre-treated with isotype control antibody (FIG. 3A), no treatment (FIG.3B), ABRAXANE® (FIG. 3C), atezolizumab (FIG. 3D), or AA130 (FIG. 3E),then labeled with fluorescently-labeled anti-PD-L1 antibody.

FIG. 4 shows the dose-dependent toxicity of ABX (solid line) and AA130(broken line) on C8161 cells.

FIGS. 5A-5D show the change in tumor volume over time in mice that wereinjected with 2×10⁶ PD-L1 positive C8161 melanoma tumor cells, thentreated by 100 ul IV tail vein injection with saline (FIG. 5A),atezolizumab alone (18 mg/kg; FIG. 5B), ABX alone (45 mg/kg; FIG. 5C)and AA130 (18 mg/kg atezolizumab and 45 mg/kg ABX; FIG. 5D) one time.Tumor growth was monitored 3 times per week. Tumor size was calculatedwith the equation: (length×width²)/2.

FIG. 6 depicts the survival of the mice from the experiment shown inFIGS. 11A-11D. Kaplan Meier curves were generated using Graph Padsoftware. The median survival for each group was 14, 13, 16, and 21.5days for saline, atezolizumab, Abraxane and AA130, respectively.Survival differences between AA130 and all other groups weresignificant, with p-values of 0.0008 for saline, 0.0015 foratezolizumab, and 0.0113 for ABX.

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications.

However, all the various embodiments of the present invention will notbe described herein. It will be understood that the embodimentspresented here are presented by way of an example only, and notlimitation. As such, this detailed description of various alternativeembodiments should not be construed to limit the scope or breadth of thepresent invention as set forth below.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Titles orsubtitles may be used in the specification for the convenience of areader, which are not intended to influence the scope of the presentinvention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In this specification and inthe claims that follow, reference will be made to a number of terms thatshall be defined to have the following meanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Preferably, the term“about” when used with regard to a dose amount means that the dose mayvary by +/−10%.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

As used herein, the term “sub-therapeutic” is used to describe an amountof antibody that is below the amount of antibody conventionally used totreat a cancer. For example, a sub-therapeutic amount is an amount lessthan that defined by the manufacturer as being required for therapy.

The term “nanoparticle” “or “nanoparticle composition” as used hereinrefers to particles having at least one dimension which is less than 5microns. In preferred embodiments, such as for intravenousadministration, the particle is less than 1 micron. For directadministration, e.g., into a tumor, the particle can be larger. Evenlarger particles are expressly contemplated by the invention.

In a population of particles, the size of individual particles aredistributed about a mean. Particle sizes for the population cantherefore be represented by an average, and also by percentiles. D50 isthe particle size below which 50% of the particles fall. 10% ofparticles are smaller than the D10 value and 90% of particles aresmaller than D90. Where unclear, the “average” size is equivalent toD50. So, for example, AA130 refers to nanoparticles having an averagesize of 130 nanometers (nm).

The term “nanoparticle” may also encompass discrete multimers of smallerunit nanoparticles. For example, a 320 nm particle comprises a dimer ofa unit 160 nm nanoparticle. For 160 nm nanoparticles, multimers wouldtherefore be approximately 320 nm, 480 nm, 640 nm, 800 nm, 960 nm, 1120nm, and so on as determined by a Mastersizer 2000 (available fromMalvern Instruments Ltd, Worcestershire, UK) as described inPCT/US15/54295.

The term “biosimilar” as used herein refers to a biopharmaceutical whichis deemed to be comparable in quality, safety, and efficacy to areference product marketed by an innovator company (Section 351(i) ofthe Public Health Service Act (42 U.S.C. 262(i)).

The term “carrier protein” as used herein refers to proteins thatfunction to transport antibodies and/or therapeutic agents. Theantibodies of the present disclosure can reversibly bind to the carrierproteins. Examples of carrier proteins are discussed in more detailbelow.

The term “core” as used herein refers to central or inner portion of thenanoparticle complex which may be comprised of a carrier protein, acarrier protein and a therapeutic agent, or other agents or combinationof agents. In some embodiments, the antibody may be non-covalentlyassociated (complexed) with the core.

As used herein, the term “enhancing the therapeutic outcome” and thelike relative to a cancer patient refers to a slowing or diminution ofthe growth of cancer cells or a solid tumor, or a reduction in the totalnumber of cancer cells or total tumor burden.

The term “therapeutic agent” as used herein means an agent which istherapeutically useful, e.g., an agent for the treatment, remission orattenuation of a disease state, physiological condition, symptoms, oretiological factors, or for the evaluation or diagnosis thereof. Atherapeutic agent may be a chemotherapeutic agent, for example, mitoticinhibitors, topoisomerase inhibitors, steroids, anti-tumor antibiotics,antimetabolites, alkylating agents, enzymes, proteasome inhibitors, orany combination thereof.

As used herein, the term, “binding agent”, “binding agent specific for”,or “binding agent that specifically binds” refers to an agent that bindsto a target antigen and does not significantly bind to unrelatedcompounds. Preferably the binding agent binds to the target antigen withhigh specificity having a dissociation constant (Kd) of 10⁻⁷ M, 10⁻⁸ M,or 10⁻⁹ M or lower. Preferably the dissociation constant is about 10⁻⁷Mto about 10⁻¹⁴ M. Examples of binding agents that can be effectivelyemployed in the disclosed methods include, but are not limited to,lectins, proteins, and antibodies, such as monoclonal antibodies, e.g.,humanized monoclonal antibodies, chimeric antibodies, or polyclonalantibodies, or antigen-binding fragments thereof, as well as aptamers,fusion proteins, and aptamers having or fused to an albumin-bindingmotif. In an embodiment the binding agent is an exogenous antibody. Anexogenous antibody is an antibody not naturally produced in a mammal,e.g., in a human, by the mammalian immune system.

As used herein, the term, “anti-PD-L1 binding agent”, “anti-PD-L1binding agent specific for,” or “anti-PD-L1 binding agent thatspecifically binds” refers to an agent that binds to a PD-L1 and doesnot significantly bind to unrelated compounds. Preferably the PD-L1binding agent binds to PD-L1 with high specificity having a dissociationconstant of 10⁻⁷ M, 10⁻⁸ M, or 10⁻⁹ M or lower. Preferably thedissociation constant is about 10⁻⁷ M to about 10⁻¹⁴ M. Examples ofanti-PD-L1 binding agents that can be effectively employed in thedisclosed methods include, but are not limited to, antibodies, such asmonoclonal antibodies, e.g., humanized monoclonal antibodies, chimericantibodies, or polyclonal antibodies, or antigen-binding fragmentsthereof, as well as aptamers, fusion proteins, and aptamers. Preferably,the binding agent has or is fused to an albumin-binding motif. In anembodiment the anti-PD-L1 binding agent is an exogenous antibody.

The term “antibody” or “antibodies” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules (i.e., molecules that contain an antigenbinding site that immuno-specifically bind an antigen). The term alsorefers to antibodies comprised of two immunoglobulin heavy chains andtwo immunoglobulin light chains as well as a variety of forms includingfull length antibodies and portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof, bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,Immunology, Benjamin, N.Y., 2ND ed. (1984); Harlow and Lane, Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapillerand Hood, Nature, 323, 15-16 (1986), which are incorporated herein byreference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE orIgD). Preferably, the antibody is IgG. An antibody may be non-human(e.g., from mouse, goat, or any other animal), fully human, humanized,or chimeric. In an embodiment the antibody is an exogenous antibody. Anexogenous antibody is an antibody not naturally produced in a mammal,e.g., in a human, by the mammalian immune system.

The term “dissociation constant,” also referred to as “Kd,” refers to aquantity expressing the extent to which a particular substance separatesinto individual components (e.g., the protein carrier, antibody, and/ortherapeutic agent).

The terms “lyophilized,” “lyophilization” and the like as used hereinrefer to a process by which the material (e.g., nanoparticles) to bedried is first frozen and then the ice or frozen solvent is removed bysublimation in a vacuum environment. An excipient is optionally includedin pre-lyophilized formulations to enhance stability of the lyophilizedproduct upon storage. In some embodiments, the nanoparticle complexescan be formed from lyophilized components (carrier protein, antibody andtherapeutic) prior to use as a therapeutic. In other embodiments, thecarrier protein, antibody, and therapeutic agent are first combined intonanoparticle complexes and then lyophilized. The lyophilized sample mayfurther contain additional excipients.

The term “buffer” encompasses those agents which maintain the solutionpH in an acceptable range prior to lyophilization and may includesuccinate (sodium or potassium), histidine, phosphate (sodium orpotassium), Tris(tris(hydroxymethyl)aminomethane), diethanolamine,citrate (sodium) and the like. In some embodiments, the buffer of thisinvention has a pH in the range from about 5.5 to about 6.5; andpreferably has a pH of about 6.0. Examples of buffers that will controlthe pH in this range include succinate (such as sodium succinate),gluconate, histidine, citrate and other organic acid buffers.

The term “pharmaceutical formulation” refers to preparations which arein such form as to permit the active ingredients to be effective, andwhich contains no additional components which are toxic to the subjectsto which the formulation would be administered.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed.

The term “reconstitution time” is the time that is required to rehydratea lyophilized formulation into a solution.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage.

The term “epitope” as used herein refers to the portion of an antigenwhich is recognized by an antibody. Epitopes include, but are notlimited to, a short amino acid sequence or peptide (optionallyglycosylated or otherwise modified) enabling a specific interaction witha protein (e.g., an antibody) or ligand. For example, an epitope may bea part of a molecule to which the antigen-binding site of an antibodyattaches.

The term “treating” or “treatment” covers the treatment of a disease ordisorder (e.g., cancer), in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisease or disorder; (iii) slowing progression of the disease ordisorder; and/or (iv) inhibiting, relieving, or slowing progression ofone or more symptoms of the disease or disorder. In some embodiments“treating” or “treatment” refers to the killing of cancer cells.

The term “kill” with respect to a cancer treatment is directed toinclude any type of manipulation that will lead to the death of thatcancer cell or at least a portion of a population of cancer cells.

The term “dose” refers to an amount of the antibody or nanoparticlecomplex given to a patient in need thereof. The attending clinician willselect an appropriate dose from a range based, e.g., on the patient'sweight, age, health, stage of cancer, level of circulating PD-L1, andother relevant factors, all of which are well within the skill of theart.

The term “unit dose” refers to a dose of the antibody or nanoparticlecomplex that is given to the patient to provide a desired result. Insome instances, the unit dose is sold in a sub-therapeutic formulation(e.g., 10% the therapeutic dose). The unit dose may be administered as asingle dose or a series of subdoses. The therapeutic dose for anantibody for a given FDA-approved indication is recited in theprescribing information, for example the therapeutic dose ofAtezolizumab, which is approved for the treatment of patients withlocally advanced or metastatic urothelial carcinoma, is 1200 mgadministered as an intravenous infusion over 60 or 30 minutes every 3weeks until disease progression or unacceptable toxicity, and preferablya subtherapeutic dose ranges from 5% to 20% of the therapeutic dose. Insuch a preferred embodiment such a subtherapeutic dose would range from60 mg/kg to 240 mg/kg, more preferably from 120 mg/kg to 180 mg/kg. Thetherapeutic dose for an antibody for a given indication where theantibody is not yet FDA approved or the antibody is not yet approved forthat indication, will be the amount the correlates to the therapeuticdose that has been approved for other indications, and thus thesubtherapeutic dose for the non-FDA approved indications is readilycalculated as a percent of the therapeutic dose (e.g., 10% of thetherapeutic dose). For example, the therapeutic dose and therefore thesubtherapeutic dose of an antibody for the treatment of metastaticmelanoma correlates to the therapeutic dose for metastatic cancers ingeneral that has been approved.

Additionally, some terms used in this specification are morespecifically defined below.

Overview

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure relates to methods for treating a patient havingcancer cells that express PD-L1, and particularly cancer cells that areor have become resistant to treatment with anti-PD-L1 antibodyimmunotherapy, by treating the patient with carrierprotein/paclitaxel/anti-PD-L1 antibody nanoparticle complexes containinga therapeutically effective amount of the paclitaxel.

The present disclosure also relates to methods for treating a patienthaving cancer cells that express PD-L1, and particularly cancer cellsthat are or have become resistant to treatment with anti-PD-L1 antibodyimmunotherapy, by treating the patient with a sub-therapeutic amount ofan anti-PD-L1 antibody and carrier protein/paclitaxel/anti-PD-L1antibody nanoparticle complexes containing a therapeutically effectiveamount of the paclitaxel.

Anti-PD-L1 Antibodies

In some embodiments, the anti-PD-L1 antibody is atezolizumab or abiosimilar version thereof.

Atezolizumab (TECENTRIQ™, Roche, USA) is a fully humanized, Fc-modifiedmonoclonal antibody of IgG1 isotype against PD-L1. Atezolizumab is aPD-L1 blocking antibody has been approved for the treatment of patientswith locally advanced or metastatic urothelial carcinoma (includingbladder cancer) and non-small cell lung cancer.

Other anti-PD-L1 antibodies are also known in the art, e.g.,BMS-936559/MDX-1105 (Bristol Myers Squibb), MeDI4736 (Durvalumab,MedImmune/AstraZeneca), and MSB00100718C (avelumab, EMD Serono).

In some embodiments, the sub-therapeutic amount of anti-PD-L1 antibodyis selected from an amount consisting of about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55% or about 60% of the therapeutic dosage ofanti-PD-L1 antibody.

In some embodiments, the sub-therapeutic amount of anti-PD-L1 antibodyis an amount which preferentially blocks circulating PD-L1 withoutblocking PD-L1 associated with the tumor.

Complexes

Methods suitable for preparing carrier protein/paclitaxel/anti-PD-L1antibody, complexes are described, for example, in U.S. Provisional App.No. 62/060,484, filed Oct. 6, 2014; and U.S. Provisional PatentApplication Nos. 62/206,770; 62/206,771; and 62/206,772 filed Aug. 18,2015, as well as PCT Publication Nos. WO2016/057554, filed Oct. 6, 2015;and WO2014/055415, filed Sep. 30, 2013. The contents of each of theseapplications are specifically incorporated by reference in theirentireties. Example 1 below provides one example of a detailed protocolfor making such complexes.

The nanoparticle complexes that may be used in the methods describedherein may also comprise a PD-L1 binding agent other than an anti-PD-L1antibody. Such PD-L1 binding agent comprises a PD-L1 binding portion andan albumin-binding motif, wherein the PD-L1 binding agent complexes witha nanoparticle of carrier protein-bound chemotherapeutic, e.g., analbumin bound paclitaxel nanoparticle, e.g., ABRAXANE®, forming ananoparticle complex that retains the ability to bind to PD-L1, e.g.,after lyophilization and reconstitution. For example that PD-L1 bindingagent may be a PD-L1-binding aptamer having or fused to analbumin-binding motif, etc.

In some embodiments, the anti-PD-L1 antibody is atezolizumab or abiosimilar version thereof. In some embodiments, the antibodies are asubstantially single layer of antibodies on all or part of the surfaceof the nanoparticle. In some embodiments the nanoparticle complexescomprise between about 100 and 1000 antibodies, or between about 400 andabout 800 antibodies.

In some embodiments the carrier protein is albumin, e.g., a human serumalbumin. In some embodiments that albumin is a recombinant human serumalbumin.

In some embodiments, the complexes further comprise at least oneadditional chemotherapeutic agent, e.g., a chemotherapeutic agentselected from the group consisting of abiraterone, bendamustine,bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil,dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide,everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib,lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone,nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin,romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide,triplatin, vinblastine, vinorelbine, vincristine, and cyclophosphamide.

In some embodiments, the carrier-bound chemotherapeutic is analbumin-bound paclitaxel, e.g., ABRAXANE® (Celgene).

In one embodiment, the antibodies of the nanoparticle complexes areintegrated onto and/or into the nanoparticle complexes, e.g. on thesurface of an albumin-bound paclitaxel core. In one embodiment, theantibodies of the nanoparticle complexes are arranged on a surface ofthe carrier protein (e.g., albumin)-bound paclitaxel core. In oneembodiment, the antibodies of the nanoparticle complexes are associatedwith the albumin-bound paclitaxel core. In one embodiment, theantibodies of the nanoparticle complexes are non-covalently associatedwith (bound to) a carrier protein, e.g. albumin, in the nanoparticlecomplex. In one embodiment, the carrier protein (e.g., albumin) andpaclitaxel are associated (bound to each other) via non-covalent bonds.

In some embodiments of the invention, the composition comprising thenanoparticle complexes may further comprise an additionalchemotherapeutic agent. The additional chemotherapeutic agent may bee.g., an alkylating agent, e.g., a platinum compound, e.g., carboplatin.

In some embodiments, the nanoparticle complex sizes are between 0.09 mto 0.9 m, between 90 nm and 800 nm, including about 90 nm, 100 nm, 130nm, 160 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm or 800 nm. Insome embodiments, the nanoparticle complex sizes are between about 100nm and about 225 nm. In other embodiments, the nanoparticle complexesare larger, e.g., from greater than 800 nm to about 3.5 μm. In someembodiments, the particles are multimers of nanoparticle complexes. Insome embodiments the nanoparticle complexes have average particle sizesof about 100 nm to about 225 nm, either freshly made or afterlyophilization and resuspension in an aqueous solution suitable forinjection.

Without being bound by theory, the binding agent is believed to be boundby the carrier protein through hydrophobic interactions, which, by theirnature, are weak. Yet the activity of the individual components, as wellas their relative relationship in the nanoparticle are preserved despitelyophilization and reconstitution of the composition. It is stillfurther contemplated that binding to the carrier protein, e.g.,complexation of the binding agent to the carrier protein, occurs throughan albumin binding motif on the binding agent, and/or anantibody-binding motif on the carrier protein. Albumin-binding motifsand antibody-binding motifs are described in PCT Application No.PCT/US17/45643, filed Aug. 4, 2017, which is incorporated herein byreference in its entirety. In some embodiments, the binding agent is anon-therapeutic and non-endogenous human antibody, a fusion protein,e.g., fusion of an antibody Fc domain to a peptide that binds a targetantigen, or an aptamer.

Treatment Methods

In one aspect is provided a method for treating a patient having acancer which expresses PD-L1, the method comprising administering to thepatient a therapeutically effective amount ofanti-PD-L1/albumin/paclitaxel nanoparticles to treat the cancer. In oneembodiment, the method comprises selecting a patient having a cancerwhich expresses PD-L1. In one embodiment, the method comprises selectinga patient having a cancer which expresses PD-L1 and is resistant totreatment with a checkpoint inhibitor immunotherapy. In one embodiment,the checkpoint inhibitor immunotherapy comprises anti-PD-L1 antibodiesthat are not part of a nanoparticle complex as described herein.

In one aspect is provided a method for treating a patient in needthereof, wherein the patient is treated with a sub-therapeutic amount ofan anti-PD-L1 antibody and albumin/paclitaxel/anti-PD-L1 antibodynanoparticle complexes containing a therapeutically effective amount ofpaclitaxel, such that the administration of the sub-therapeutic amountof the anti-PD-L1 antibody enhances the efficacy of the nanoparticlecomplexes. A subject in need thereof may be a subject afflicted with acancer wherein the cancer cells express or over express PD-L1. Thesubject may also be afflicted with a cancer wherein the cancer cellsexpress or over express PD-L1 but are resistant to treatment with ananti-PD-L1 antibody immunotherapy. In one embodiment, thesub-therapeutic amount of anti-PD-L1 antibodies is not in a nanoparticlecomplex comprising a carrier protein (e.g., albumin) boundchemotherapeutic (“uncomplexed” anti-PD-L1 antibody).

In one embodiment, the method comprises selecting a patient having acancer wherein the cancer cells express or over express PD-L1. In oneembodiment, the method comprises selecting a patient having a cancerwherein the cancer cells express or overexpress PD-L1 but are resistantto treatment with an anti-PD-L1 antibody immunotherapy. Methods areknown in the art for determining whether a tumor comprises cancer cellsexpressing PD-L1, e.g., the Ventana PD-L1 (sp263) Assay (Roche), whichwas approved by the FDA as a complementary diagnostic to provide PD-L1status on patients with metastatic urothelial cancer, and the PD-L1 IHC28-8 pharmDx assay (Dako, Agilent Pathology Solutions).

In some embodiments of this invention, the nanoparticle complexescomprise a PD-L1 binding agent other than an anti-PD-L1 antibody. Insome embodiments of this invention, the nanoparticle complexes comprisean anti-PD-L1 antibody that is the same antibody as the “uncomplexed”anti-PD-L1 antibody. In some embodiments of this invention, thenanoparticle complexes comprise an anti-PD-L1 antibody that is adifferent antibody than the “uncomplexed” anti-PD-L1 antibody.

The patient may be co-treated with a sub-therapeutic amount of ananti-PD-L1 antibody and carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody nanoparticle complex.

For the sake of clarification, “co-treatment” refers to treatment of thecancer expressing PD-L1 with an anti-PD-L1 antibody prior to,concurrently with, or immediately after administration of the carrier(e.g., albumin)/paclitaxel/anti-PD-L1 antibody nanoparticle complexes,such that the anti-PD-L1 antibody is capable of binding circulatingPD-L1.

In one embodiment, the anti-PD-L1 antibody is administered in asub-therapeutic dose prior to administration of the nanoparticlecomplexes. In this embodiment, the administration of the anti-PD-L1antibody occurs about 0.5 hours to about 48 hours prior toadministration of the nanoparticle complexes.

In another embodiment, the anti-PD-L1 antibody composition isadministered between 0.5 hours prior to and up to 0.5 hours afteradministration of the nanoparticle complexes. In this embodiment, it iscontemplated that such administration will nevertheless result inbinding of some of the circulating PD-L1 by the antibody.

In yet another embodiment, the antibody composition can be administeredup to 2 hours post administration of the nanoparticle complexes.

In a preferred aspect, there is provided methods for enhancing theefficacy of albumin/paclitaxel/anti-PD-L1 antibody nanoparticlecomplexes by administering the albumin/paclitaxel/anti-PD-L1 antibodynanoparticle complexes about 0.5 hours to 48 hours after pretreatment ofa patient with a sub-therapeutic amount of anti-PD-L1 antibody.Preferably, such nanoparticle complexes are administered about 24 hoursafter the sub-therapeutic amount of anti-PD-L1 antibody.

In another aspect, there is provided methods for enhancing thetherapeutic outcome in a patient suffering from a cancer expressingPD-L1 which patient is selected to be treated with nanoparticlescomprising albumin, paclitaxel and anti-PD-L1 antibodies, which methodcomprises treating the patient with a sub-therapeutic amount of theanti-PD-L1 antibody prior to any subsequent treatment with thenanoparticles.

In another aspect, there is provided methods for enhancing thetherapeutic outcome in a patient suffering from a cancer overexpressingPD-L1, the method comprising co-treating the patient with asub-therapeutic amount of the anti-PD-L1 antibody and with an effectiveamount of nanoparticle complexes comprising albumin-bound paclitaxel andanti-PD-L1 antibodies.

In another aspect, there is provided a method for enhancing thetherapeutic outcome in a patient suffering from a cancer expressingPD-L1, which patient is to be treated with nanoparticle complexescomprising albumin, paclitaxel and anti-PD-L1 antibodies, which methodcomprises treating the patient with a sub-therapeutic amount of theanti-PD-L1 antibody within +/−0.5 hours of administration of thenanoparticles.

In another aspect is provided a method for enhancing the therapeuticoutcome in a patient suffering from a cancer overexpressing PD-L1 whichpatient has been treated with a sub-therapeutic amount of the anti-PD-L1antibody, the method comprising treating the patients with an effectiveamount of nanoparticles comprising albumin-bound paclitaxel andanti-PD-L1 antibodies within +/−0.5 hours of administration of theantibodies.

In some embodiments the anti-PD-L1 antibody is administered prior to thecarrier protein (e.g., albumin)/paclitaxel/anti-PD-L1 antibody complex,for example, the anti-PD-L1 antibody can be administered minutes, hoursor days prior to administration of the carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody complex. In some embodiments,the anti-PD-L1 antibody is administered between about 5 to about 59minutes, about 10 to about 50 minutes, about 15 to about 45 minutes,about 20 to about 40 minutes, about 25 to about 35 minutes prior toadministration of the carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody nanoparticle complex. In otherembodiments, the anti-PD-L1 antibody can be administered about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours,or longer prior to administration of the carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody complex. In other embodiments,the anti-PD-L1 antibody can be administered about 1 day, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days, about 7 days,about 10 days, about 12 days, about 15 days, or longer prior toadministration of the carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody complex. Contemplated valuesinclude any value, subrange, or range within any of the recited rangesor values, including endpoints.

In some embodiments, the anti-PD-L1 antibody can be administeredconcurrently with administration of the carrier protein (e.g.,albumin)/paclitaxel/anti-PD-L1 antibody complex, for example, within 10minutes or less of each other.

In other embodiments, the anti-PD-L1 antibody can be administeredsubsequent to administration of the albumin/paclitaxel/anti-PD-L1antibody complex, for example, within 2 hours after administration ofthe albumin/paclitaxel/anti-PD-L1 antibody complex.

Cancers or tumors that can be treated by the compositions and methodsdescribed herein include, but are not limited to: biliary tract cancer;brain cancer, including glioblastomas and medulloblastomas; breastcancer; cervical cancer; choriocarcinoma; colon cancer; endometrialcancer; esophageal cancer, gastric cancer; hematological neoplasms,including acute lymphocytic and myelogenous leukemia; multiple myeloma;AIDS associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms, including Bowen's disease and Paget'sdisease; liver cancer (hepatocarcinoma); lung cancer; lymphomas,including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas;oral cancer, including squamous cell carcinoma; ovarian cancer,including those arising from epithelial cells, stromal cells, germ cellsand mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. Cancers suitable fortreatment with the methods described herein include but are not limitedto cancers that express or overexpress PD-L1. Cancers that may betreated with the methods disclosed herein also include cancers that areresistant to treatment with anti-PD-L1 antibody immunotherapy, includingcancers that had been responsive to immunotherapy but developedresistance to the anti-PD-L1 antibody immunotherapy, which anti-PD-L1antibodies are not in complex with a nanoparticle comprising a carrierprotein (e.g., albumin)-bound chemotherapeutic (e.g., ABRAXANE®).

Antibody Formulations

In one aspect, the anti-PD-L1 antibody is a unit-dose formulation of ananti-PD-L1 antibody which formulation comprises from about 1% to about60% of a therapeutic dose of the antibody, wherein the formulation ispackaged so as to be administered as a unit dose. In an aspect of theinvention, the unit-dose formulation of an anti-PD-L1 antibody comprisesabout 10% of a therapeutic dose of the antibody. For example 10% of atherapeutic dose of an anti-PD-L1 antibody, e.g., atezolizumab, may be60 mg to 240 mg.

The unit-dose formulation of an anti-PD-L1 antibody can be about 1% toabout 60%, about 5% to about 50%, about 10% to about 40%, about 15% toabout 30%, about 20% to about 25%, of a therapeutic dose of theanti-PD-L1 antibody. Contemplated values include any value, subrange, orrange within any of the recited ranges, including endpoints.

In some embodiments, the anti-PD-L1 antibody is atezolizumab or abiosimilar version thereof, which formulation comprises from about 5% toabout 20% of a therapeutic dose of atezolizumab or a biosimilar versionthereof.

In another aspect, provided herein is a formulation comprising ananti-PD-L1 antibody provided herein, and at least one pharmaceuticallyacceptable excipient.

In general, the unit-dose formulations provided herein can be formulatedfor administration to a patient by any of the accepted modes ofadministration. Various formulations and drug delivery systems areavailable in the art. See, e.g., Remington's Pharmaceutical Sciences,edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

In general, unit-dose formulations provided herein will be administeredas pharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration.

The unit-dose formulations may be comprised of, in general, ananti-PD-L1 antibody, optionally in combination with at least onepharmaceutically acceptable excipient. Acceptable excipients arenon-toxic, aid administration, and do not adversely affect thetherapeutic benefit of the claimed compounds. Such excipient may be anysolid, liquid, semisolid or, in the case of an aerosol composition,gaseous excipient that is generally available to one of skill in theart.

Nanoparticle Complex Formulations

In one aspect, the composition comprising the nanoparticle complexesdescribed herein is formulated for systemic delivery, e.g., intravenousadministration.

In one aspect, the nanoparticle composition is formulated for directinjection into a tumor. Direct injection includes injection into orproximal to a tumor site, perfusion into a tumor, and the like. Becausethe nanoparticle composition is not administered systemically, ananoparticle composition is formulated for direct injection into a tumormay comprise any average particle size. Without being bound by theory,it is believed that larger particles (e.g., greater than 500 nm, greaterthan 1 μm, and the like) are more likely to be immobilized within thetumor, thereby providing what is believed to be a better beneficialeffect.

In another aspect, provided herein is a composition comprising acompound provided herein, and at least one pharmaceutically acceptableexcipient.

In general, the compounds provided herein can be formulated foradministration to a patient by any of the accepted modes ofadministration. Various formulations and drug delivery systems areavailable in the art. See, e.g., Gennaro, A. R., ed. (1995) Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co.

In general, compounds provided herein will be administered aspharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration.

The formulations described herein may include excipients. Solidpharmaceutical excipients include starch, cellulose, talc, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Othersuitable pharmaceutical excipients and their formulations are describedin Remington's Pharmaceutical Sciences, edited by E. W. Martin (MackPublishing Company, 18th ed., 1990).

The present formulations may, if desired, be presented in a pack ordispenser device containing a unit-dose of the active ingredient. Such apack or device may, for example, comprise metal or plastic foil, such asa blister pack, or glass, and rubber stoppers such as in vials. The packor dispenser device may be accompanied by instructions foradministration. Compositions comprising a unit-dose formulation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Kits

In some aspects, the current invention relates to kits comprising: (a)an amount of albumin-/paclitaxel/anti-PD-L1 antibody nanoparticlecomplexes, (b) a unit dose of a sub-therapeutic amount of anti-PD-L1antibody, and optionally (c) instructions for use.

In some embodiments, the kits can include lyophilized complexes of thealbumin/paclitaxel/anti-PD-L1 antibody.

In some preferred embodiments, the kit components can be configured insuch a way that the components are accessed in their order of use. Forexample, in some aspects the kit can be configured such that uponopening or being accessed by a user, the first component available isthe unit dose of a sub-therapeutic amount of anti-PD-L1 antibody, forexample, in a first vial. A second container (e.g., a vial) comprisingor containing an amount of the albumin/paclitaxel/anti-PD-L1 antibodynanoparticle complexes can then be accessed. As such the kits can beintuitively configured in a way such that the first vial must be openedprior to the second vial being opened. It should be understood that insome embodiments, the order can be different, for example, where it isdesired to administer the complex first, prior to the administration ofthe antibody. Also, it can be configured such that both are administeredat the same time. Finally, it should be understood that additional vialsor containers of either or both component(s) can be included, andconfigured for opening in any desired order. For example, the first vialcould be antibody, the second vial could include complex, a third couldinclude either antibody or complex, etc. It is contemplated that a kitconfigured in such a way would prevent, or at least help to prevent, thecomponents from being administered in an order not intended by theinstructions for use.

In some aspects, the invention is directed to a kit of parts foradministration of albumin/paclitaxel/anti-PD-L1 antibody complexes and aunit dose of a sub-therapeutic amount of anti-PD-L1 antibody; andoptionally further comprising a dosing treatment schedule in a readablemedium. In some embodiments, the dosing schedule includes thesub-therapeutic amount of anti-PD-L1 antibody required to achieve adesired average serum level is provided. In some embodiments, the kit ofparts includes a dosing schedule that provides an attending clinicianthe ability to select a dosing regimen of the sub-therapeutic amount ofanti-PD-L1 antibody based on the sex of the patient, mass of thepatient, and the serum level that the clinician desires to achieve. Insome embodiments, the dosing treatment is based on the level ofcirculating PD-L1 in the blood of the patient. In some embodiments, thedosing schedule further provides information corresponding to the volumeof blood in a patient based upon weight (or mass) and sex of thepatient. In an embodiment, the storage medium can include anaccompanying pamphlet or similar written information that accompaniesthe unit dose form in the kit. In an embodiment, the storage medium caninclude electronic, optical, or other data storage, such as anon-volatile memory, for example, to store a digitally-encodedmachine-readable representation of such information.

The term “readable medium” as used herein refers to a representation ofdata that can be read, for example, by a human or by a machine.Non-limiting examples of human-readable formats include pamphlets,inserts, or other written forms. Non-limiting examples ofmachine-readable formats include any mechanism that provides (i.e.,stores and/or transmits) information in a form readable by a machine(e.g., a computer, tablet, and/or smartphone). For example, amachine-readable medium includes read-only memory (ROM); random accessmemory (RAM); magnetic disk storage media; optical storage media; andflash memory devices. In one embodiment, the machine-readable medium isa CD-ROM. In one embodiment, the machine-readable medium is a USB drive.In one embodiment, the machine-readable medium is a Quick Response Code(QR Code) or other matrix barcode.

EXAMPLES

The present disclosure is illustrated using a pre-treatment ofatezolizumab (i.e., TECENTRIQ™) followed by nanoparticles composed ofalbumin-bound paclitaxel (i.e., ABRAXANE®) and atezolizumab (i.e.,TECENTRIQ™).

One skilled in the art would understand that making and using thenanoparticles, as well as administration of a co-treatment ofatezolizumab, of the Examples are for the sole purpose of illustration,and that the present disclosure is not limited by this illustration.

Any abbreviation used herein, has normal scientific meaning. Alltemperatures are ° C. unless otherwise stated. Herein, the followingterms have the following meanings unless otherwise defined:

ABX = ABRAXANE ®/(albumin-bound paclitaxel) ATZ = atezolizumab BSA =bovine serum albumin kg = kilogram nM = nano molar mg milligram ml or mL= milliliter m² = square meters mm³ = cubic millimeter μg = microgram μl= microliter μm = micrometer/micron PBS = Phosphate buffered saline

Example 1: Making Atezolizumab-ABRAXANE® Nanoparticles

Atezolizumab and ABRAXANE® (ABX) were co-incubated at room temperaturefor 30 minutes at a concentration of 4 mg/mL and 10 mg/mL, respectivelyto form the nanoparticle, AA130.

To determine whether atezolizumab and ABX are capable of interacting toform nanoparticle complexes, Biolayer interferometry (BLItz) (ForteBioscience) was performed using streptavidin probes. 100 ug/ml ofbiotinylated atezolizumab in 1×PBS was bound to the streptavidin probe.After washing unbound atezolizumab from the probe, the antibody-boundprobe was exposed to ABX at concentrations of 100, 500, 1000 μg/mL in1×PBS. An antibody probe exposed to PBS was used as background andbackground was subtracted. BLItz software was used to calculatedissociation constants (FIG. 1). The Kd was determined to be 1.462×10⁻⁹.

Example 2: Size Determination of Atezolizumab-ABRAXANE® Nanoparticles

Mastersizer NS300 was employed to determine the particle size ofatezolizumab bound ABX relative to ABX alone. Nanosight uses dynamiclight scattering and Brownian motion to calculate particle size.

Atezolizumab and ABX were co-incubated to form the nanoparticle, AA130,as described above. ABX was diluted 1:200 and atezolizumab-bound ABX wasdiluted 1:800; three 30-second video clips were captured and analyzed todetermine particle size (FIG. 2A). FIG. 2B is a still image from one ofthe video clips of AA130. The average particle size of theatezolizumab-ABX nanoparticles was determined to be about 129 nm;average size of ABX alone is about 90 nm.

Example 3: AA130 Binds PD-L1

Flow cytometry was performed to access binding of atezolizumab andatezolizumab bound Abraxane to the ligand, PD-L1. The PD-L1 positivemelanoma cell line, C8161 was used for this experiment. AA130 was madeas described above and an aliquot of the nanoparticles was spun at 6000rpm for 10 minutes to remove any unbound atezolizumab. C8161 cells werestained with FITC labeled isotype control and anti-human PD-L1 asnegative and positive controls, respectively. The C8161 cells wereincubated for 30 minutes with ABX and atezolizumab alone and the AA130nanoparticle. After the incubation the cells were labeled with FITClabeled anti-human PD-L1 for 30 minutes and washed with FACS buffer(1×PBS+0.5% BSA and 0.05% Na azide). After washing, the cells wereanalyzed by flow cytometer on the Guava 8HT and data analysis performedwith Gauvasoft software (Millipore).

C8161 cells were pre-treated with isotype control antibody (FIG. 3A), notreatment (FIG. 3B), ABRAXANE® (FIG. 3C), atezolizumab (FIG. 3D), orAA130 (FIG. 3E), then labeled with fluorescently-labeled anti-PD-L1antibody. The atezolizumab in the context of the 130 nm particle retainsits ability to bind its ligand, PD-L1.

Example 4: AA130 Cellular Toxicity

C8161 melanoma cells were exposed to ABX and AA130 at paclitaxelconcentrations from 0 to 200 μg/mL overnight to determine cell toxicity.The cells were also incubated with EdU, a thymidine analog. The next daythe cells were harvested, fixed with 2% paraformaldehyde andpermeabolized with 1% saponin. After permeabolization the cells wereincubated for 30 minutes with a FITC labeled anti-EdU antibody todetermine the percentage of cells proliferating. After washing, thecells were analyzed by flow cytometer on the Guava 8HT and data analysisperformed with Gauvasoft software (Millipore). The proliferation indexwas calculated by normalization to an untreated positive control.

FIG. 4 shows the dose-dependent toxicity of ABX (solid line) and AA130(broken line) on C8161 cells. The AA130 nanoparticle complex hascellular toxicity similar to ABX alone.

Example 5: Making of Intravenous Formulation of AA130 NanoparticleComplexes

For use in humans, the AA130 complexes are prepared by obtaining thedose appropriate number of 4 mL vials of 25 mg/mL ATZ and diluting eachvial per the following directions to 4 mg/mL. The dose appropriatenumber of 100 mg vials of ABX is prepared by reconstituting to a finalconcentration containing 10 mg/mL ABX nanoparticles. Using a sterile 3mL syringe, 1.6 mL (40 mg) of atezolizumab (25 mg/mL) is withdrawn andslowly injected, over a minimum of 1 minute, onto the inside wall ofeach of the vials containing 100 mg of ABX. The atezolizumab solutionshould not be injected directly onto the lyophilized cake as this willresult in foaming. Then, using a sterile 12 mL sterile syringe, 8.4 mL0.9% Sodium Chloride Injection, USP, is withdrawn and slowly injected,over a minimum of 1 minute, 8.4 mL onto the inside wall of each vialcontaining ABX 100 mg and ATZ 40 mg. Once the addition of ATZ 1.6 mL and0.9% Sodium Chloride Injection, USP 8.4 mL is completed, each vial isgently swirled and/or inverted slowly for at least 2 minutes untilcomplete dissolution of any cake/powder occurs. Generation of foamshould be avoided. At this point, the concentration of each vial shouldbe 100 mg/10 mL ABX and 40 mg/10 mL ATZ. The vials containing the ABXand ATZ should sit for 60 minutes. The vial(s) is gently swirled and/orinverted every 10 minutes to continue to mix the complex. After 60minutes has elapsed, the calculated dosing volume of ABX and ATZ iswithdrawn from each vial and is slowly added to an empty viaflex bag. Anequal volume of 0.9% Sodium Chloride Injection, USP is then added tomake the final concentration of ABX 5 mg/mL and ATZ 2 mg/mL. The bag isthen be gently swirled and/or inverted slowly for 1 minute to mix. TheABX:ATZ nanoparticles are stored for up to 4 hours at room temperaturefollowing final dilution.

Example 6: Co-Treatment with ATZ Improves Targeting of ABX/ATZ Complexes

Athymic nude mice are injected with 1×10⁶ A375 human melanoma cells inthe right flank and then are treated with PBS, 12 mg/kg ATZ, 30 mg/kgABX, AA130, or pretreated with 1.2 mg/kg ATZ and, 24 hr later, AA130.AA130 are prepared as described in PCT Application No. PCT/US15/54295and Example 1 above. It is contemplated that only mice treated withAA130 (with or without pretreatment with ATZ) will show reduction inaverage tumor volume.

It is also contemplated that pretreatment with ATZ will be associatedwith a statistically significant reduction in tumor volume over controlor ATZ alone, or ABX alone.

Tumors are measured on day 15 following treatment with either saline(PBS), TECENTRIQ™ (ATZ), ABRAXANE® (ABX), AA130, or a pretreatment ofATZ one day before AA130 (ATZ+AA130). A 10% sub-therapeutic dose of ATZ,as compared to the dose give to the ATZ alone or AA130 cohort, is givento the ATZ+AA130 cohort 24 hours prior to administration of the AA130.It is contemplated that the ATZ+AA130 cohort will present with delayedtumor growth, even when compared to AA130. It is contemplated that theseexperiments will show that pre-treatment with ATZ+AA130, increasessurvival.

Survival is again assessed at day 40. It is contemplated that mediansurvival of mice treated with ATZ pretreatment and AA130 will exceedmedian survival or the mice treated with either PBS or ATZ alone.

Example 7: Fluorescence Over Time of AlexaFluor 750 LabeledNanoparticles

Mice are injected IV with equal amounts of either labeled ABRAXANE®, ornanoparticles of ABRAXANE® having surface complexation with atezolizumab(ATZ) as per Example 1 above (AA130); one AA130 group of mice receives apre-treatment of 1.2 mg/kg atezolizumab. Fluorescent imagery isperformed at an excitation/emission spectrum of 710/760. Regions ofinterest (ROI) in the mice are assigned by software to track tumoraccumulation based on a fluorescence threshold. Fluorescence per unitarea of background ROIs and tumor ROIs for all three treatment groups isdetermined at 24, 29, and 48 hours post injection

The amount of fluorescence (and thus paclitaxel) in the tumor andbackground ROIs at 24, 29 and 48 hour are determined and it iscontemplated that the data will demonstrate that pretreatment with ATZresults in higher levels of tumor fluorescence as compared AA130 aloneor ABRAXANE alone. It is contemplated that pretreatment with ATZ and useof ABRAXANE® nanoparticles having surface complexation with ATZ providesfor a method for increasing the duration of tumor uptake of albumincontaining a chemotherapeutic agent both at 24 hours and 48 hours. It isalso contemplated that use of ABRAXANE® nanoparticles having surfacecomplexation with ATZ also provides for increasing the duration of tumoruptake of these albumin containing nanoparticles with or withoutpretreatment with ATZ at 48 hours.

Without being limited to any theory, the antibody coating of the albuminnanoparticles imparts stability possibly by reducing liver or kidneyclearance and/or by reducing protease degradation of the albumincarrier.

Example 8: In Vivo Efficacy of AA130 Nanoparticles

Athymic nude mice (Harlan Sprague Dawley) were injected with 2×10⁶ PD-L1positive C8161 melanoma tumor cells. The tumors were allowed to growuntil about 600 mm³ and were treated by 100 μl IV tail vein injectionwith saline, atezolizumab alone (18 mg/kg), ABX alone (45 mg/kg) andAA130 (18 mg/kg atezolizumab and 45 mg/kg ABX) one time (FIGS. 5A-5D).Tumor growth was monitored 3 times/week. Tumor size was calculated withthe equation: (length×width²)/2.

Tumor growth curves (FIG. 6) show slowed tumor growth in the micetreated with AA130 relative to saline and the individual drugs alone.Kaplan Meier curves were generated using Graph Pad software. The mediansurvival for each group was 14, 13, 16, and 21.5 days for saline,atezolizumab, ABX and AA130, respectively. Survival differences betweenAA130 and all other groups were significant with p-values of 0.0008 forsaline, 0.0015 for atezolizumab, and 0.0113 for Abraxane.

1-68. (canceled)
 69. A unit-dose formulation of an anti-PD-L1 antibody which formulation comprises from about 1% to about 60% of a therapeutic dose of said antibody, wherein said formulation is packaged so as to be administered as a unit dose.
 70. The unit-dose formulation of claim 69, wherein the anti-PD-L1 antibody is atezolizumab or a biosimilar version thereof.
 71. The unit-dose formulation of claim 69 which formulation comprises from about 5% to about 20% of a therapeutic dose of atezolizumab or a biosimilar version thereof.
 72. A kit comprising: (a) an amount of an albumin/paclitaxel/anti-PD-L1 antibody nanoparticle complexes, (b) a unit dose of a sub-therapeutic amount of anti-PD-L1 antibody, and optionally (c) instructions for use.
 73. The kit of claim 72, wherein the albumin/paclitaxel/anti-PD-L1 antibody nanoparticle complexes are lyophilized. 